Remedies for tumor in hematopoietic organs

ABSTRACT

An inducing agent or enhancing agent, for the expression of HM1.24 antigen in hematopoietic tumor cells, comprising interferon α, interferon γ, or the IRF-2 protein as an active ingredient, as well as an anti-tumor agent for hematopoietic tumors which comprises a combination of said inducing agent or enhancing agent and an antibody against HM1.24.

FIELD OF THE INVENTION

The present invention relates to the use of interferon α, interferon γ,and the IRF-2 protein as expression-enhancing agents for HM1.24 antigenin myeloma.

The present invention also relates to the use of interferon α,interferon γ, and the IRF-2 protein as expression-enhancing agents forHM1.24 antigen in lymphatic tumors. Furthermore, the present inventionrelates to a method of treating leukemia using anti-HM1.24 antibody andinterferon α or interferon γ.

BACKGROUND ART

Leukocytes occurring in normal human peripheral blood comprisegranulocytes, monocytes and lymphocytes, and granulocytes are furtherdivided into neutrophils, eosinophils, and basophils. In the productionof these blood cells, myelocytic stem cells and lymphatic stem cellsdifferentiate from common undifferentiated hematopoietic stem cells, andfrom these stem cells, finally, each line of leukocytes differentiates.Blood cell-related cells including these hematopoietic stem cells arealso referred to as hematopoietic cells. Tumors (hematopoietic tumors)of hematopoietic cells include leukemia, lymphoma, myeloma, and thelike.

Leukemia is a disease of cancerated hematopoietic cells, in which bonemarrow is occupied by leukemic cells and thereby normal hematopoieticfunctions are inhibited, resulting in the decreased production of normalblood cells and in subsequent development of anemia, leukopenia andthrombocytopenia. Also, based on the origin of leukemic cells, leukemiais roughly divided into two types: myelocytic leukemia and lymphocyticleukemia, each of which is further divided into the acute form and thechronic form. Furthermore, as a subtype, mixed lineage leukemia havingcellular traits of the two lineages, bone marrow lineage and lymphocytelineage, is also known.

Tumorigenesis takes place at the level of hematopoietic stem cells,wherein there are a case in which differentiation stops at a certainstage of differentiation and maturation and tumors are only formed inthe cells upstream thereof, and a case in which the functions ofdifferentiation and maturation are retained though it evades thebiological regulatory functions and exhibits autonomous growth. Theformer includes acute leukemia, and the latter includes chronic leukemiaand the myelodysplastic syndrome. Based on the identity of the growingcells, acute leukemia is roughly divided into acute myelocytic leukemia(AML), acute monocytic leukemia (AMoL), acute erythroleukemia,megakaryobalastic leukemia, and acute lymphocytic leukemia (ALL).

As a subtype, acute promyelocytic leukemia (APL) is known. Acuteleukemia and the myelodysplastic syndrome may be classified based on theFrench-American-British classification (FAB classification). In the FABclassification, acute lymphocytic leukemia is divided into L1, L2, andL3, and for example Burkitt's lymphoma is classified into L3. Acutemyelocytic leukemia is divided into M0, M1, M2, M3, M4, M5, M6, and M7and, for example, erythrocyte abnormality is classified into M6 andmegakaryobalastic leukemia is classified into M7. These methods ofclassification and of testing are known and are described in manytextbooks (for example, Shin-Rinsho Naikagaku (New Clinical InternalMedicine), Fumimaro Takaku and Etsuro Ogata, Igakushoin Ltd., 1999).

Also, based on the identity of growing cells, chronic leukemia isroughly divided into chronic myelocytic leukemia (CML) and chroniclymphocytic leukemia (CLL). Also, as a subtype of chronic myelocyticleukemia, chronic myelomonocytic leukemia is known, and as a subtype ofchronic lymphocytic leukemia, prolymphocytic leukemia is known.

Lymphoma is a generic term for tumors derived from cells constitutinglymphatic tissues such as the lymph node, and is hematopoietic celltumors caused mainly by canceration of lymphocytes. Malignant lymphomais divided into Hodgkin's disease and non-Hodgkin lymphoma, both ofwhich are cancerated lymphocytic cells and can be divided into the Tlymphocytic and the B lymphocytic types.

As non-Hodgkin lymphoma, there are known B lymphocytic tumors such asfollicular lymphoma, mantle cell lymphoma, Burkitt's lymphoma, pre-Blymphoma and the like. For T lymphocytic tumors, there are known adult Tcell leukemia (ATL) and peripheral non-ATL T-lymphoma (PNTL). Also,diffuse lymphoma comprising two types (T lymphocytic and B lymphocytic)is included in non-Hodgkin lymphoma. Furthermore, as a subtype, hairycell leukemia which is a B lymphocytic tumor is known.

Lymphocytic leukemia and lymphoma which are canceration of majorconstituent cells of lymphocytes are referred to as lymphocytic tumors,and are roughly divided into B lymphocytic tumors and T lymphocytictumors. For example, B lymphocytic tumors include acute B lymphocyticleukemia (B-ALL), chronic B lymphocytic leukemia (B-CLL), pre-Blymphoma, Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma,diffuse lymphoma and the like. T lymphocytic tumors include acute Tlymphocytic leukemia (T-ALL), chronic T lymphocytic leukemia (T-CLL),adult T cell leukemia (ATL), peripheral non-ATL T-lymphoma (PNTL) andthe like (Zukai Rinshogan series (Illustrated Clinical Cancer Series)No. 17, Leukemia and lymphoma, Takashi Sugimura et al., MEDICAL VIEWCo., Ltd., 1987; B Lymphocytic Tumors, Kiyoshi Takatsuki, NishimuraCo.,Ltd., 1991; Shin-Rinsho Naikagaku (New Clinical Internal Medicine),Fumimaro Takaku and Etsuro Ogata, Igakushoin Ltd., 1999). Myeloma isalso a type of lymphatic tumor, and exhibits characteristic clinicalfindings.

Myeloma which is also referred to as plasmacytoma and multiple myelomais a neoplastic disease characterized by the accumulation of monoclonalplasma cells in the bone marrow. Myeloma is a disease in which plasmacells, i.e. terminally differentiated B cells that produce and secreteimmunoglobulins, are monoclonally increased mainly in the bone marrow,and thus in the serum of patients with this disease, monoclonalimmunoglobulins or components thereof, L chain, H chain, etc., can bedetected.

For the treatment of myeloma, chemotherapeutic agents etc. have beenused so far, but no effective therapeutic agents have been found thatlead to the complete remission and the extension of the survival ofpatients. Thus, there has been a long-awaited need for agents havingtherapeutic effects based on a new mechanism of action. For lymphoma andleukemia as well, though moderately effective chemotherapy has beendeveloped, new agents have been waited for due to adverse reactions.

On the other hand, Goto, T. et al. have reported a monoclonal antibody(mouse anti-HM1.24 antibody) that was obtained by immunizing mice withhuman plasma cells (Blood (1994) 84, 1922-1930). When anti-HM1.24antibody was administered to a mouse transplanted with human myelomacells, the antibody accumulated in tumor tissues in a specific manner(Masaaki Kosaka et al., Nippon Rinsho (Japan Clinical) (1995) 53,627-635), suggesting that anti-HM1.24 antibody could be applied in thediagnosis of tumor localization by radioisotopic labeling, missiletherapies such as radiotherapy, and the like.

In the above Blood (1994) 84, 1922-1930, it has been described thatanti-HM1.24 antibody has an in vitro cytotoxicity on a human myelomacell line RPMI8226. It has also been shown that chimeric anti-HM1.24antibody, or anti-HM1.24 antibody that is mouse anti-HM1.24 antibodyrendered chimeric, and a humanized reshaped anti-HM1.24 antibody,specifically bind to myeloma cells and have cytotoxicity (Blood (1999)93, 3922-3920).

On the other hand, it has also been demonstrated for lymphocytic tumorsthat an antigen protein recognized by anti-HM1.24 antibody is expressedin lymphocytic tumors, and that anti-HM1.24 antibody has a cytotoxicityon lymphocytic tumors due to a CDC activity and an ADCC activity, andthereby exhibits anti-tumor effect (WO 98/35698). Thus, HM1.24 antigenhas been highly expressed not only in myeloma cells that are terminallydifferentiated B cells but also in lymphocytic tumors, and anti-HM1.24antibody that recognizes HM1.24 antigen is useful as a therapeutic agentfor lymphocytic tumors.

Thus, HM1.24 antigen has been highly expressed in myeloma cells that areterminally differentiated B cells and in lymphocytic tumors, andanti-HM1.24 antibody that recognizes this antigen exhibits cell-killingactivity in proportion to the number of HM1.24 antigens on the cellsurface, and thus immunotherapy with anti-HM1.24 antibody is expected toprovide an effective method of treating multiple myeloma and lymphocytictumors. Thus, if the amount of HM1.24 antigen, which is an antigenagainst anti-HM1.24 antibody, expressed on the cell surface could beenhanced, the administration of a smaller amount of the antibody isexpected to provide equivalent cytotoxicity, and it would becomepossible to decrease adverse reactions.

Furthermore, for hematopoietic tumor cells that are not expressingHM1.24 antigen, if the amount of HM1.24 antigen expressed on the cellsurface could be enhanced, cytotoxicity or cytocidal effect through ADCCactivity or CDC activity with anti-HM1.24 antibody is expected forhematopoietic tumor cells for which, generally, anti-HM1.24 antibodyalone is not effective.

On the other hand, interferon, that was discovered as a substance havingan activity of inhibiting viral growth, is currently known to beclassified into four groups of α, β, γ, and {overscore (ω)} in mammals,and to have a variety of biological activities (Pestka, S., et al., Ann.Rev. Biochem. (1987) 56, 727-777; Langer, J. A., et al., ImmunologyToday (1988) 9, 393-400). However, there were no reports on whetherinterferon α and interferon γ could have an effect of increasing theexpressed amount of HM1.24 antigen in myeloma cells or cells ofhematopoietic tumors such as lymphocytic tumors.

On the other hand, interferon regulatory factor (IRF)-1 and 2 wereidentified as a transcription regulatory factor of the IFN-β gene. IRF-1and 2 are generally known to bind to the same gene regulatory sequence,and act in an antagonistic manner in that IRF-1 acs as a transcriptionactivation factor and IRF-2 as a transcription suppressing factor. TheNIH3T3 cells in which IRF-2 was highly expressed has been demonstratedto exhibit enhanced cell saturation density, colony formation in themethylcellulose gel, and a tumorigenic property in nude mice, and IRF-2has been shown to act as an oncogene.

On the other hand, recent advances in research have indicated that IRF-2is required for the expression of histone H4 that acts in the control ofcell cycle. IRF-2 has also been shown to increase the expression ofvascular cell adhesion molecule-1 (VCAM-1) in muscle cells, and it hasalso been demonstrated that the acid region (182 to 218) of IRF-2 isinvolved in the activation of VCAM-1. Based on this, it is known thatIRF-2 not only acts as a transcription regulatory factor but may act asa transcription activation factor.

However, it was not known that the IRF-2 protein binds to the promoter(HM1.24 promoter) of the HM1.24 antigen gene, and activates saidpromoter.

DISCLOSURE OF THE INVENTION

Current methods of treating myeloma are, as mentioned above, notsatisfactory and, accordingly, the appearance of epoch-makingtherapeutic drugs or methods that prolong the patient's survival isawaited. The treatment of myeloma with anti-HM1.24 antibody is likely toprovide epoch-making therapeutic drugs in terms of specificity andeffectiveness, and thus there is a need for methods of allowinganti-HM1.24 antibody to exhibit its effect more efficiently.

Also, as methods of treating lymphocytic tumors currently employed,there can be mentioned various chemotherapies, X-ray therapy, bonemarrow transplantation and the like, but no therapeutic methods aresatisfactory, and thus epoch-making therapeutic drugs or methods thatattains complete remission of lymphocytic tumors and prolong thepatient's survival are awaited. Furthermore, therapeutic methods thatare effective for leukemia other than lymphocytic leukemia, i.e.myelocytic leukemia such as acute myelocytic leukemia and chroniclymphocytic leukemia are awaited. Therapeutic drugs and methods thatattain complete remission of these myelomas, lymphocytic tumors, andmyelocytic leukemia and that can prolong the patient's survival couldprovide therapeutic drugs and methods for hematopoietic tumors ingeneral.

Thus, it is an object of the present invention to provide a means ofenhancing the effect of anti-HM1.24 antibody of suppressing myeloma byenhancing the amount expressed of HM1.24 antigen in myeloma.

It is another object of the present invention to provide a means ofenhancing the effect of anti-HM1.24 antibody of suppressing lymphocytictumors by enhancing the amount expressed of HM1.24 antigen inlymphocytic tumors.

It is further object of the present invention to provide a means ofinducing the effect of anti-HM1.24 antibody of suppressing myelocyticleukemia by providing a means of newly inducing the expression of HM1.24antigen in myelocytic leukemia.

The above should provide a means of treating hematopoietic tumors withanti-HM1.24 antibody by enhancing the amount expressed of HM1.24 antigenor by using a means of newly inducing HM1.24 antigen.

After searching drugs that either enhance the amount expressed of HM1.24antigen or allow. HM1.24 antigen to be newly expressed on the cellsurface, the present inventors have found that interferon α, interferonγ, and the IRF-2 protein have the desired activity, and thereby havecompleted the present invention.

Thus the present invention provides an expression-enhancing orexpression-inducing agent of a protein (HM1.24 antigen) having the aminoacid sequence set forth in SEQ ID NO: 2 in hematopoietic tumor cells,said agent comprising interferon α, interferon γ, or the IRF-2 proteinas an active ingredient.

The above hematopoietic tumors are, for example, leukemia, lymphoma,myeloma etc., and as this leukemia includes, for example, acutemyelocytic leukemia, chronic myelocytic leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia etc., the above lymphomaincludes, for example, Hodgkin's disease, T lymphocytic non-Hodgkinlymphoma, B lymphocytic non-Hodgkin lymphoma etc., and the above myelomaincludes multiple myeloma.

The present inventors have also found that, by allowing an antigen thatspecifically binds to said HM1.24 antigen to bind to the hematopoietictumor cells in which HM1.24 antigen has been expressed by the aboveexpression-enhancing or expression-inducing agent of HM1.24 antigen,anti-tumor effect on said hematopoietic tumor cells can be enhanced.

Thus, the present invention also provides a therapeutic agent or apharmaceutical composition for the treatment of hematopoietic tumorscomprising, as an active ingredient, an antibody that specifically bindsto a protein having the amino acid sequence set forth in SEQ ID NO: 2,wherein interferon α, interferon γ, or the IRF-2 protein is used incombination.

The present invention also provides a therapeutic agent or apharmaceutical composition for the treatment of hematopoietic tumorscomprising, as an active ingredient, (1) interferon α, interferon γ, orthe IRF-2 protein, and (2) an antibody that specifically binds to aprotein having the amino acid sequence set forth in SEQ ID NO: 2.

The present invention also provides a therapeutic agent or apharmaceutical composition for the treatment of hematopoietic tumorscomprising, as an active ingredient, interferon α, interferon γ, or theIRF-2 protein, wherein an antibody that specifically binds to a proteinhaving the amino acid sequence set forth in SEQ ID NO: 2 is used incombination.

The above hematopoietic tumors are, for example, leukemia, lymphoma, ormyeloma. The above leukemia includes, for example, acute myelocyticleukemia, chronic myelocytic leukemia, acute lymphocytic leukemia,chronic lymphocytic leukemia etc., the above lymphoma includes, forexample, Hodgkin's disease, T lymphocytic non-Hodgkin lymphoma, Blymphocytic non-Hodgkin lymphoma etc., and the above myeloma includesmultiple myeloma.

The above antibody is preferably an antibody having cytotoxicity, andthe cytotoxicity is the ADCC activity. The antibody is preferably amonoclonal antibody, a chimeric antibody, a humanized antibody, or ahuman antibody. The monoclonal antibody is preferably anti-HM1.24antibody produced by a hybridoma having the Deposit No. FERM BP-5233,and said chimeric antibody or humanized antibody is preferably achimeric antibody or a humanized antibody of anti-HM1.24 antibodyproduced by a hybridoma having the Deposit No. FERM BP-5233.

The present invention further provides a kit for the treatment of apatient having a hematopoietic tumor, said kit comprising:

(1) an antibody that specifically binds to a protein having the aminoacid sequence set forth in SEQ ID NO: 2; and

(2) an instruction manual instructing the administration of the aboveantibody to the patient in combination with an expression-enhancingagent of a protein having the amino acid sequence set forth in SEQ IDNO: 2.

The present invention also provides a kit for the treatment of a patienthaving a hematopoietic tumor, said kit comprising:

(1) an expression-enhancing agent of a protein having the amino acidsequence set forth in SEQ ID NO: 2; and

(2) an instruction manual instructing the administration of the aboveantibody to the patient in combination with an antibody thatspecifically binds to a protein having the amino acid sequence set forthin SEQ ID NO: 2.

The present invention also provides a kit for the treatment of a patienthaving a hematopoietic tumor, said kit comprising:

(1) an expression-enhancing agent of a protein having the amino acidsequence set forth in SEQ ID NO: 2; and

(2) an antibody that specifically binds to a protein having the aminoacid sequence set forth in SEQ ID NO: 2; and

(3) an instruction manual instructing the combined administration of theabove agent and the above antibody to the patient.

The present invention also provides a method of screening anexpression-enhancing agent of HM1.24 antigen, comprising the steps of:

(1) preparing cells having a reporter gene that has the region of theHM1.24 gene promoter;

(2) contacting said cells with a test substance; and

(3) detecting the expression of the reporter gene.

The present invention also provides an expression-enhancing agent ofHM1.24 antigen selected by the above method.

The present invention also provides a pharmaceutical composition for thetreatment of hematopoietic tumors wherein said composition comprises theabove expression-enhancing agent and said composition is used incombination with an antibody that specifically binds to a protein havingthe amino acid sequence set forth in SEQ ID NO: 2.

The present invention further provides a method of screening anexpression-enhancing agent comprising the steps of:

(1) contacting cells with a test substance;

(2) determining the amount expressed of the IL-2 protein in said cells.

The present invention also provides an expression-enhancing agent of theIRF-2 protein selected by the above method.

The present invention also provides a pharmaceutical composition for thetreatment of hematopoietic tumors wherein said composition comprises theabove expression-enhancing agent and said composition is used incombination with an antibody that specifically binds to a protein havingthe amino acid sequence set forth in SEQ ID NO: 2.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows the result of an experiment in which a myeloma cell lineU266 cultured in the absence (upper) or the presence (bottom) ofinterferon α was analyzed by flow cytometry using human IgG (control) oranti-HM1.24 antibody as a label.

FIG. 2 shows the result of an experiment in which the myeloma cells of apatient cultured in the absence (upper) or the presence (bottom) ofinterferon α was analyzed by flow cytometry using human IgG (control) oranti-HM1.24 antibody as a label.

FIG. 3 is a graph showing the result of an experiment in which U266cells transformed with a reporter plasmid into which the promoter regionof a gene encoding HM1.24 antigen had been inserted were cultured in theabsence or in the presence of various concentrations of interferon α,and then the luciferase activity was determined.

FIG. 4 is a graph showing the result of an experiment in which U266cells or HEL cells transformed with a reporter plasmid into which asegment from the transcription initiation point to 151 bp upstream or to77 bp upstream among the promoter region of a gene encoding HM1.24antigen had been inserted were cultured in the presence of interferon α(1000 U/ml), and then the luciferase activity was determined.

FIG. 5 shows the result of an experiment in which a myeloma cell lineU266 cultured in the absence (upper) or the presence (bottom) ofinterferon γ was analyzed by flow cytometry using human IgG (control) oranti-HM1.24 antibody as a label.

FIG. 6 shows the result of an experiment in which the myeloma cells ofthe patient cultured in the absence (upper) or the presence (bottom) ofinterferon γ were analyzed by flow cytometry using human IgG (control)or anti-HM1.24 antibody as a label.

FIG. 7 is an electrophoretogram that shows changes with time in theamount of a transcription factor that is produced by adding IFN a tocultured U266 cells and that binds to the HM1.24 promoter region, and isa photograph substituting for a drawing. NE(−): no nuclear extract wasadded. Oh: the nuclear extract without IFN-α stimulation was added.0.5-8 h: the nuclear extracts that passed respective time afterstimulation with IFN-α (1000 U/ml) was added. + cold: 50 ng of thenonlabeled ISRE2 probe was added. + cold unrelated: 50 ng of thenonlabeled adp sequence was added.

FIG. 8 is an electrophoretogram showing the result of an experiment inwhich a transcription factor that binds to HM1.24 promoter wasidentified using various antibodies, and is a photograph substitutingfor a drawing. NE(−): no nuclear extract was added. Oh: the nuclearextract without IFN-α stimulation was added. 8 h: the nuclear extract 8hours after stimulation with IFN-α (1000 U/ml) was added. + cold: 50 ngof the nonlabeled ISRE2 probe was added. + cold unrelated: 50 ng of thenonlabeled adp sequence was added. Two μg of each antibody was added.

FIG. 9 is a graph showing the result of an experiment in which theHM1.24 promoter reporter plasmid and the IRF-2 expression plasmid wereintroduced into U266 cells, and the reporter activity was determined.

FIG. 10 shows the result of an experiment in which a human leukemia cellline HEL and cells collected from a patient with acute myelocyticleukemia were cultured in the absence (left) or the presence (right) ofinterferon α, and then were analyzed by flow cytometry using human IgG(control) or anti-HM1.24 antibody as a label. The dark area representsthe control and the light area represents the stain with anti-HM1.24antibody.

FIG. 11 shows the result of an experiment in which cells collected froma patient with acute lymphocytic leukemia and a patient with Blymphocytic non-Hodgkin lymphoma were cultured in the absence (left) orthe presence (right) of interferon α, and then were analyzed by flowcytometry using human IgG (control) or anti-HM1.24 antibody as a label.The dark area represents the control and the light area represents thestain with anti-HM1.24 antibody.

FIG. 12 shows the result of an experiment in which cells collected froma patient with T lymphocytic non-Hodgkin lymphoma were cultured in theabsence (left) or the presence (right) of interferon α, and then wereanalyzed by flow cytometry using human IgG (control) or anti-HM1.24antibody as a label. The dark area represents the control and the lightarea represents the stain with anti-HM1.24 antibody.

FIG. 13 shows the result of an experiment in which ADCC activity byvarious human anti-HM1.24 antibodies were determined when peripheralblood mononuclear cells from normal healthy subjects were used as theeffector cell using a human leukemia cell line HEL as the standard cell.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Interferon-α and Interferon-γ

Interferon was discovered as a substance having an activity ofinhibiting viral growth and currently four types, α, β, γ, and{overscore (ω)}, are known in mammals. In addition to the activity ofinhibiting viral growth, they are known to exhibit an activity ofinhibiting cell growth and of modulating immunological functions(Interferon “Cytokine”, Toshiaki Osawa etd., (1990) 115-133, TokyoKagaku Dojin Co., Ltd.; Pestka, S., et al., Ann. Rev. Biochem. (1987)56, 727-777; Langer, J. A., et al., Immunology Today (1988) 9, 393-400).

Interferon-α and interferon-γ for use in the present invention may bemutants as long as they have an activity of increasing the amountexpressed of HM1.24 antigen. In order to determine the amount expressedof HM1.24 antigen, as described in Examples, myeloma cells are harvestedfrom a myeloma cell line or a patient with myeloma and then subjected toflow cytometry for detection. As mutants, they may be interferon-α andinterferon-γ in which one or several, or a plurality of amino acidresidues have been deleted, substituted, or substituted or inserted.

As methods of introducing deletion, substitution, or insertion intoproteins, there can be used site-directed mutagenesis that alters thecorresponding gene (Hashimoto-Gotoh, Gene (1005) 152, 271-275, Zoller,Methods Enzymol. (1983) 100, 468-500, Kramer, Nucleic Acids Res. (1984)12, 9441-8456, Kunkel, Proc. Natl. Acad. Sci. USA (1985) 82, 489-492,“New Cell Engineering Experimental Protocol” edited by Dept. ofOncology, Inst. of Medical Science, Univ. of Tokyo (1993) pp. 241-248).

It is also possible to use “Site-Directed Mutagenesis System”(GIBCO-BRL) and “QuickChange Site-Directed Mutagenesis Kit” (Stratagene)employing commercially available PCR. Amino acid mutations in proteinsmay sometimes take place in nature. That such a protein, in whichmutation has been introduced, has an activity equal to the originalprotein has been shown in Mark, Proc. Natl. Acad. Sci. USA (1984) 81,5662-5666.

In the substitution of amino acid residues, it is preferred tosubstitute between amino acids whose properties are conserved. Forexample, substitution is preferred between hydrophobic amino acids (A,I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G,H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I,P), amino acids having hydroxyl group-containing side chains (S, T, Y),amino acids having sulfur-containing side chains (C, M), amino acidshaving carboxylic acid- and amide-containing side chains (D, N, E, Q),amino acids having base-containing side chains (R, K, H), and aminoacids having aromatic group-containing side chains (H, F, Y, W).

Furthermore, as mutants, peptide fragments of interferon-α orinterferon-γ may be used. In particular, peptide fragments that havebinding sites with interferon-α or interferon-γ receptors are preferred.Preferably they are peptides comprised of 100 or more, more preferably130 or more, still more preferably 150, and most preferably 160 or morecontiguous amino acid residues.

IRF-2 Protein

Interferon regulatory factors (IRF)-1 and -2 were identified astranscription regulatory factors of the IFN-β gene (Taniguchi, T. etal., Nucleic Acids Res. (1989) 17, 8372; Taniguchi, T. et al., Cell(1989) 58, 729). IRF-1 and -2 are generally known to bind to the samegene regulatory sequence: IRF-1 and IRF-2 act in an antagonistic mannerin that IRF-1 acs as a transcription activation factor, whereas IRF-2 asa transcription suppressing factor. The NIH3T3 cells in which IRF-2 ishighly expressed has been demonstrated to exhibit enhanced cellsaturation density, colony formation in the methylcellulose gel, and atumorigenic property in nude mice, and IRF-2 acts as an oncogene.

On the other hand, recent advances in research have indicated that IRF-2is required for the expression of histone H4 that acts for the controlof cell cycle. IRF-2 is also shown to increase the expression ofvascular cell adhesion molecule-1 (VCAM-1) in muscle cells, and it isbecoming increasingly clear that the acid region (182 to 218) of IRF-2is involved in the activation of VCAM-1. Based on this, it is known thatIRF-2 not only acts as a transcription regulatory factor but as atranscription activation factor.

Hybridoma

The hybridoma that produces the antibody for use in the presentinvention can be basically constructed using a known technology asdescribed below. Thus, the HM1.24 antigen protein or a HM1.24antigen-expressing cell may be used as a sensitizing antigen and isimmunized in the conventional method of immunization. The immune cellsthus obtained are fused with known parent cells in the conventional cellfusion process, and then monoclonal antibody-producing cells arescreened by the conventional screening method to construct the desiredhybridoma.

Specifically, monoclonal antibody may be obtained in the followingmanner. For example, as the HM1.24 antigen-expressing cell which is thesensitizing antigen to obtain antibody, a human multiple myeloma cellline KPMM2 (Japanese Unexamined Patent Publication (Kokai) No. 7-236475)and KPC-32 (Goto, T. et al., Jpn. J. Clin. Hematol. (1991) 32, 1400) canbe used. As the sensitizing antigen, it is also possible to use aprotein having the amino acid sequence set forth in SEQ ID NO: 1 or apeptide or a polypeptide containing an epitope recognized by anti-HM1.24antibody.

The CDNA of the protein having the amino acid sequence set forth in SEQID NO 1 used as the sensitizing antigen may be inserted into the XbaIcleavage site of the pUC19 vector to prepare a plasmid pRS38-pUC19. E.coli having the plasmid pRS38-pUC19 has been internationally depositedunder the provisions of the Budapest Treaty as Escherichia coli DH5α(pRS38-pUC19) on Oct. 5, 1993 with the Patent Microorganism Depository,the National Institute of Bioscience and Human Technology (Chuo Dai 6,1-1, Higashi 1-chome, Tsukuba city, Ibaraki Pref., Japan) as FERMBP-4434 (Japanese Unexamined Patent Publication (Kokai) No. 7-196694).Using the cDNA fragment contained in this plasmid pRS38-pUC19, a peptideor a polypeptide that contains an epitope recognized by anti-HM1.24antibody can be constructed by gene engineering technology.

Mammals to be immunized with the sensitizing antigen are notspecifically limited, and they are preferably selected in considerationof their compatibility with the parent cell for use in cell fusion. Theygenerally include rodents such as mice, rats, and hamsters.

Immunization of animals with a sensitizing antigen may be carried outusing a known method. A general method, for example, involves theintraperitoneal or subcutaneous administration of a sensitizing antigento the mammal.

Specifically, a sensitizing antigen which has been diluted and suspendedin an appropriate amount of phosphate buffered saline (PBS) orphysiological saline etc. is mixed, as desired, with an appropriateamount of a common adjuvant, for example Freund's complete adjuvant.After being emulsified, it is preferably administered to the mammal forseveral times every 4 to 21 days. Alternatively, a suitable carrier maybe used at the time of immunization of the sensitizing antigen.

After immunizing in this manner and confirming an increase in thedesired antibody levels in the serum, immune cells are harvested fromthe mammal and are subjected to cell fusion. As immune cells to besubjected to cell fusion, there may be specifically mentioned spleencells.

The mammalian myeloma cells as the other parent cells which aresubjected to cell fusion with the above-mentioned immune cellspreferably include various known cell lines such as P3X63Ag8.653 (J.Immunol. (1979) 123: 1548-1550), P3X63Ag8U.1 (Current Topics inMicrobiology and Immunology (1978) 81: 1-7), NS-1 (Kohler, G. andMilstein, C., Eur. J. Immunol. (1976) 6: 511-519), MPC-11 (Margulies, D.H. et al., Cell (1976) 8: 405-415), SP2/0 (Shulman, M. et al., Nature(1978) 276: 269-270), FO (de St. Groth, S. F. et al. J. Immunol. Methods(1980) 35: 1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978) 148:313-323), R210 (Galfre, G. et al., Nature (1979) 277: 131-133) and thelike.

Cell fusion between the above immune cells and the myeloma cells may beessentially conducted in accordance with a known method such as isdescribed in Milstein et al. (Kohler, G. and Milstein, C., MethodsEnzymol. (1981) 73: 3-46) and the like.

More specifically, the above cell fusion is carried out in theconventional nutrient broth in the presence of, for example, a cellfusion accelerator. As the cell fusion accelerator, for example,polyethylene glycol (PEG), Sendai virus (HVJ) and the like may be used,and, in addition, an adjuvant such as dimethyl sulfoxide etc. may beadded as desired to enhance the efficiency of the fusion.

The preferred ratio of the immune cells and the myeloma cells to be usedis, for example, 1 to 10 times more immune cells than the myeloma cells.Examples of culture media to be used for the above cell fusion includeRPMI1640 medium and MEM culture medium suitable for the growth of theabove myeloma cell lines, and the conventional culture medium used forthis type of cell culture, and besides a serum supplement such as fetalcalf serum (FCS) may be added.

In cell fusion, predetermined amounts of the above immune cells and themyeloma cells are mixed well in the above culture liquid, to which a PEGsolution previously heated to about 37° C., for example a PEG solutionwith a mean molecular weight of about 1000 to 6000, is added at aconcentration of 30 to 60% (w/v), and mixed to obtain the desired fusioncells (hybridomas). Then by repeating the sequential addition of asuitable culture liquid and centrifugation to remove the supernatant,cell fusion agents etc. which are undesirable for the growth of thehybridoma can be removed.

Said hybridoma is selected by culturing in a conventional selectionmedium, for example, the HAT culture medium (a culture liquid containinghypoxanthine, aminopterin, and thymidine). Culturing in said HAT culturemedium is continued generally for a period of time sufficient to effectkilling of the cells (non-fusion cells) other than the desiredhybridoma, generally several days to several weeks. Then, theconventional limiting dilution method is conducted in which thehybridomas that produce the desired antibody are screened andmonoclonally cloned.

In addition to obtaining the above hybridoma by immunizing an animalother than the human with an antigen, it is also possible to sensitizehuman lymphocytes in vitro with HM1.24 antigen or HM1.24antigen-expressing cells, and to allow the resulting sensitizedlymphocytes to be fused with a human myeloma cell, for example U266, andthereby to obtain the desired human antibody having the activity ofbinding to HM1.24 antigen or to HM1.24 antigen-expressing cells (seeJapanese Examined Patent Publication (Kokoku) No. 1-59878). Furthermore,a transgenic animal having a repertoire of human antibody genes isimmunized with HM1.24 antigen or HM1.24 antigen-expressing cells toobtain the desired antibody according to the above-mentioned method (seeInternational Patent Publication WO 93/12227, WO 92/03918, WO 94/02602,WO 94/25585, WO 96/34096, and WO 96/33735).

Furthermore, using a human antibody library, the desired human antibodymay be isolated by means of panning. For example, the variable region ofhuman antibody is expressed on the surface of a phage by the phagedisplay method as a single chain antibody (scFv) to select a phage thatbinds to the HM1.24 antigen using a HM1.24 antigen-immobilized plate. Byanalyzing the gene of the phage selected, the gene encoding the variableregion of the human antibody that binds to the HM1.24 antigen can beidentified. Using these gene sequences, anti-HM1.24 antibody can beprepared. These methods are already known and can be found in WO92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO95/01438, and WO 95/15388.

The monoclonal antibody-producing hybridomas thus constructed can besubcultured in the conventional culture liquid, or can be stored for aprolonged period of time in liquid nitrogen.

In order to obtain monoclonal antibodies from said hybridoma, there maybe employed a method in which said hybridoma is cultured by theconventional method and the antibodies are obtained as the culturesupernatant, or a method in which the hybridoma is administered to andgrown in a mammal compatible with said hybridoma and the antibodies areobtained as the ascites, or other methods. The former method is suitablefor obtaining high-purity antibodies, whereas the latter is suitable fora large scale production of antibodies.

Monoclonal Antibody

Specifically the anti-HM1.24 antibody-producing hybridoma can beconstructed using the method of Goto, T. et al. (Blood (1994) 84:1922-1930). It can be conducted by: a method in which the anti-HM1.24antibody-producing hybridoma that was internationally deposited underthe provisions of the Budapest Treaty as FERM BP-5233 on Apr. 27, 1995with the Patent Microorganism Depository, the National Institute ofBioscience and Human Technology (Chuo Dai 6, 1-1, Higashi 1-chome,Tsukuba city, Ibaraki Pref., Japan) is intraperitoneally injected toBALB/c mice (manufactured by CLEA Japan) to obtain the ascites, fromwhich the anti-HM1.24 antibody is purified, or: a method in which saidhybridoma is cultured in a suitable culture medium such as the RPMI1640medium containing 10% bovine fetal serum and 5% BM-Condimed H1(manufactured by Boehringer Mannheim), the hybridoma SFM medium(manufactured by GIBCO-BRL), the PFHM-II medium (manufactured byGIBCO-BRL) and the like, and the anti-HM1.24 antibody can be purifiedfrom the supernatant.

Recombinant Antibody

A recombinant antibody which was produced by the recombinant genetechnology, in which an antibody gene was cloned from the hybridoma andintegrated into a suitable vector which was then introduced into a host,can be used in the present invention as monoclonal antibody (see, forexample, Carl, A. K., Borrebaeck, and James, W. Larrick, THERAPEUTICMONOCLONAL ANTIBODIES, published in the United Kingdom by MACMILLANPUBLISHERS LTD. 1990).

Specifically, mRNA encoding the variable region (V) of the desiredantibody is isolated from the hybridoma producing the antibody. Theisolation of mRNA is conducted by preparing total RNA using, forexample, a known method such as the guanidine ultracentrifuge method(Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), the AGPCmethod (Chmczynski, P. et al., (1987) 162, 156-159), and then mRNA ispurified from the total RNA using the mRNA Purification kit(manufactured by Pharmacia) and the like. Alternatively, mRNA can bedirectly prepared using the QuickPrep mRNA Purification Kit(manufactured by Pharmacia).

cDNA of the V region of the antibody may be synthesized using a reversetranscriptase from the mRNA thus obtained. cDNA may be synthesized usingthe AMV Reverse Transcriptase First-strand cDNA Synthesis Kit and thelike. Alternatively, for the synthesis and amplification of cDNA, the5′-Ampli FINDER RACE Kit (manufactured by Clontech) and the 5′-RACEmethod (Frohman, M. A. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17,2919-2932) that employs PCR may be used. The desired DNA fragment ispurified from the PCR product obtained and may be ligated to vector DNA.Moreover, a recombinant vector is constructed therefrom and then isintroduced into E. coli etc., from which colonies are selected toprepare the desired recombinant vector. The base sequence of the desiredDNA may be confirmed by a known method such as the dideoxy method.

Once the DNA encoding the V region of the desired antibody has beenobtained, it may be ligated to DNA encoding the constant region (Cregion) of the desired antibody, which is then integrated into anexpression vector. Alternatively, the DNA encoding the V region of theantibody may be integrated into an expression vector which alreadycontains DNA encoding the C region of the antibody.

In order to produce the antibody for use in the present invention, theantibody gene is integrated as described below into an expression vectorso as to be expressed under the control of the expression regulatoryregion, for example an enhancer and/or a promoter. Subsequently, theexpression vector may be transformed into a host cell and the antibodycan then be expressed therein.

Altered Antibody

In accordance with the present invention, artificially alteredrecombinant antibody such as chimeric antibody and humanized antibodycan be used for the purpose of lowering heterologous antigenicityagainst humans. These altered antibody can be produced using knownmethods.

Chimeric antibody can be obtained by ligating the thus obtained DNAencoding the V region of antibody to DNA encoding the C region of humanantibody, which is then integrated into an expression vector andintroduced into a host for production of the antibody therein (seeEuropean Patent Application EP 125023, and International PatentPublication WO 96/02576). Using this known method, chimeric antibodyuseful for the present invention can be obtained.

For example, E. coli having the plasmid that contains the L chain andthe H chain of chimeric anti-HM1.24 antibody has been internationallydeposited under the provisions of the Budapest Treaty as Escherichiacoli DH5α (pUC19-1.24L-gκ) and Escherichia coli DH5α (pUC19-1.24H-gγ1),respectively, on Aug. 29, 1996 with the Patent Microorganism Depository,the National Institute of Bioscience and Human Technology (Chuo Dai 6,1-1, Higashi 1-chome, Tsukuba city, Ibaraki Pref., Japan) as FERMBP-5646 and FERM BP-5644, respectively (see Japanese Patent ApplicationNo. 8-264756).

Humanized antibody which is also referred to as reshaped human antibodyhas been made by transplanting the complementarity determining region(CDR) of antibody of a mammal other than the human, for example mouseantibody, into the CDR of human antibody. The general recombinant DNAtechnology for preparation of such antibodies is also known (seeEuropean Patent Application EP 125023 and International PatentPublication WO 96/02576).

Specifically, a DNA sequence which was designed to ligate the CDR ofmouse antibody with the framework region (FR) of human antibody issynthesized by the PCR method from several divided oligonucleotideshaving sections overlapping with one another at the ends thereof. TheDNA thus obtained is ligated to the DNA encoding the C region of humanantibody, and then is integrated into an expression vector, which isintroduced into a host for antibody production (see European PatentApplication EP 239400 and International Patent Publication WO 96/02576).

For the FR of human antibody ligated through CDR, the FR is selected forwhich the complementarity determining region forms a favorable antigenbinding site. When desired, amino acids in the framework region of theantibody variable region may be substituted so that the complementaritydetermining region of reshaped human antibody may form an appropriateantigen binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

For example, E. coli having the plasmid that contains the L chain andthe H chain of humanized anti-HM1.24 antibody has been internationallydeposited under the provisions of the Budapest Treaty as Escherichiacoli DH5α (pUC19-RVLa-AHM-gκ) and Escherichia coli DH5α(pUC19-RVHr-AHM-gγ1), respectively, on Aug. 29, 1996 with the PatentMicroorganism Depository, the National Institute of Bioscience and HumanTechnology (Chuo Dai 6, 1-1, Higashi 1-chome, Tsukuba city, IbarakiPref., Japan) as FERM BP-5645 and FERM BP-5643, respectively (PatentApplication No. 8-264756).

For chimeric antibody or humanized antibody, the C region of humanantibody is used and, as the C region of human antibody that exhibitscytotoxicity, human Cγ, for example Cγ1, Cγ2, Cγ3, and Cγ4, can be used.Among them, antibody having Cγ1 and Cγ3 in particular has potentcytotoxicity, i.e. ADCC activity and CDC activity, and is usedpreferably in the present invention.

Chimeric antibody consists of the variable region of antibody derivedfrom a mammal other than the human and the C region derived from humanantibody, whereas humanized antibody consists of the complementaritydetermining region of antibody derived from a mammal other than thehuman and the framework region (FR) and the C region of antibody derivedfrom human antibody. Accordingly, antigenicity thereof in the human bodyhas been reduced so that they are useful as the active ingredient of thetherapeutic agents of the present invention.

A preferred embodiment of the humanized antibody for use in the presentinvention includes humanized anti-HM1.24 antibody (see WO 98/14580).

Expression and Production

Antibody genes constructed as described above may be expressed andobtained in a known method. In the case of mammalian cells, expressionmay be accomplished using an expression vector containing a commonlyused useful promoter, the antibody gene to be expressed, and DNA inwhich the poly A signal has been operably linked at 3′ downstreamthereof or a vector containing said DNA. Examples of thepromoter/enhancer include human cytomegalovirus immediate earlypromoter/enhancer.

Additionally, as the promoter/enhancer which can be used for theexpression of antibody for use in the present invention, there can beused viral promoters/enhancers such as retrovirus, polyoma virus,adenovirus, and simian virus 40 (SV40), and promoters/enhancers derivedfrom mammalian cells such as human elongation factor 1α (HEF1α).

For example, expression may be readily accomplished by the method ofMulligan et al. (Nature (1979) 277, 108) when SV40 promoter/enhancer isused, or by the method of Mizushima et al. (Nucleic Acids Res. (1990)18, 5322) when HEF1α promoter/enhancer is used.

In the case of E. coli, expression may be conducted by operably linkinga commonly used useful promoter, a signal sequence for antibodysecretion, and the antibody gene to be expressed, followed by expressionthereof. As the promoter, for example, there can be mentioned lacZpromoter and araB promoter. The method of Ward et al. (Nature (1098)341, 544-546; FASEB J. (1992) 6, 2422-2427) may be used when laczpromoter is used, and the method of Better et al. (Science (1988) 240,1041-1043) may be used when araB promoter is used.

As the signal sequence for antibody secretion, when produced in theperiplasm of E. coli, the pelB signal sequence (Lei, S.P. et al., J.Bacteriol. (1987) 169, 4379) can be used. After separating the antibodyproduced in the periplasm, the structure of the antibody isappropriately refolded before use (see, for example, WO 96/30394).

As the origin of replication, there can be used those derived from SV40,polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like.Furthermore, for the amplification of the gene copy number in the hostcell system, expression vectors can include as selectable markers theaminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene,E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene, thedihydrofolate reductase (dhfr) gene and the like.

For the production of antibody for use in the present invention, anyproduction system can be used. The production system of antibodypreparation comprises the in vitro and the in vivo production system. Asthe in vitro production system, there can be mentioned a productionsystem which employs eukaryotic cells and the production system whichemploys prokaryotic cells.

When the eukaryotic cells are used, there are the production systemswhich employ animal cells, plant cells, and fungal cells. Known animalcells include (1) mammalian cells such as CHO cells, COS cells, myelomacells, baby hamster kidney (BHK) cells, HeLa cells, and Vero cells, (2)amphibian cells such as Xenopus oocytes, or (3) insect cells such assf9, sf21, and Tn5. Known plant cells include, for example, thosederived from the genus Nicotiana, more specifically cells derived fromNicotiana tabacum, which is subjected to callus culture. Known fungalcells include yeasts such as the genus Saccharomyces, more specificallySaccharomyces cereviceae, or filamentous fungi such as the genusAspergillus, more specifically Aspergillus niger.

When the prokaryotic cells are used, there are the production systemswhich employ bacterial cells. Known bacterial cells include Escherichiacoli (E. coli), and Bacillus subtilis.

By introducing, via transformation, the gene of the desired antibodyinto these cells and culturing the transformed cells in vitro, theantibody can be obtained. Culturing is conducted in the known methods.For example, as the culture liquid, DMEM, MEM, RPMI1640, and IMDM can beused, and serum supplements such as fetal calf serum (FCS) may be usedin combination. In addition, antibodies may be produced in vivo byimplanting cells into which the antibody gene has been introduced intothe abdominal cavity of an animal and the like.

As further in vivo production systems, there can be mentioned thosewhich employ animals and those which employ plants. When animals areused, there are the production systems which employ mammals and insects.

As mammals, goats, pigs, sheep, mice, and cattle can be used (VickiGlaser, SPECTRUM Biotechnology Applications, 1993). As insects,silkworms can be used.

When plants are used, tabacco, for example, can be used.

Antibody genes are introduced into these animals or plants, and theantibodies are produced in such animals or plants, and recoveredtherefrom. For example, an antibody gene is inserted into the middle ofthe gene encoding protein which is inherently produced in the milk suchas goat β casein to prepare fusion genes. DNA fragments containing thefusion gene into which the antibody gene has been inserted are injectedinto a goat embryo, and the embryo is introduced into a female goat. Thedesired antibody is obtained from the milk produced by the transgenicgoat born to the goat who received the embryo or offsprings thereof. Inorder to increase the amount of milk containing the desired antibodyproduced by the transgenic goat, hormones may be given to the transgenicgoat as appropriate. (Ebert, K. M. et al., Bio/Technology (1994) 12,699-702).

When silkworms are used, baculovirus into which the desired antibodygene has been inserted is infected to the silkworm, and the desiredantibody can be obtained from the body fluid of the silkworm (Susumu, M.et al., Nature (1985) 315, 592-594). Moreover, when tabacco is used, thedesired antibody gene is inserted into an expression vector for plants,for example pMON 530, and then the vector is introduced into a bacteriumsuch as Agrobacterium tumefaciens. The bacterium is then infected totabacco such as Nicotiana tabacum to obtain the desired antibody fromthe leaves of the tabacco (Julian, K.-C. Ma et al., Eur. J. Immunol.(1994) 24, 131-138).

When antibody is produced in the in vitro or in vivo production systems,as described above, DNA encoding the heavy chain (H chain) or the lightchain (L chain) of antibody may be separately integrated into anexpression vector and the hosts are transformed simultaneously, or DNAencoding the H chain and the L chain may be integrated into a singleexpression vector and the host is transformed therewith (seeInternational Patent Publication WO 94-11523).

The antibody produced as described above can be bound to variousmolecules such as polyethylene glycol (PEG) for use as a modifiedantibody. “Antibody” as used herein includes these modified antibodies.In order to obtain these modified antibody, the antibody obtained may bechemically modified. These methods have already been established in thefield of the art.

Separation and Purification of Antibody

Antibodies produced and expressed as described above can be separatedfrom the inside or outside of the cell or from the host and then may bepurified to homogeneity. Separation and purification of the antibody foruse in the present invention may be accomplished by affinitychromatography. As the column used for such affinity chromatography,there can be mentioned Protein A column and Protein G column. Examplesof the column employing Protein A column are Hyper D, POROS, SepharoseF. F. and the like.

Alternatively, methods for separation and purification conventionallyused for proteins can be used without any limitation. Separation andpurification of the antibody for use in the present invention may beaccomplished by combining, as appropriate, chromatography other than theabove-mentioned affinity chromatography, filtration, ultrafiltration,salting-out, dialysis and the like. Chromatography includes, forexample, ion exchange chromatography, hydrophobic chromatography,gel-filtration and the like.

Determination of Antibody Concentration

The concentration of antibody obtained in the above method can bedetermined by the measurement of absorbance or by ELISA and the like.Thus, when absorbance measurement is employed, the antibody for use inthe present invention or a sample containing the antibody isappropriately diluted with PBS(−) and then the absorbance is measured at280 nm, followed by calculation using the absorption coefficient of 1.35OD at 1 mg/ml. When the ELISA method is used, measurement is conductedas follows. Thus, 100 μl of goat anti-human IgG (manufactured by BIOSOURCE) diluted to 1 μg/ml in 0.1 M bicarbonate buffer, pH 9.6, is addedto a 96-well plate (manufactured by Nunc), and is incubated overnight at4° C. to immobilize the antibody.

After blocking, 100 ml each of appropriately diluted antibody of thepresent invention or a sample containing the antibody, or 100 ml ofhuman IgG (manufactured by CAPPEL) as the standard is added, andincubated at room temperature for 1 hour. After washing, 100 l of5000-fold diluted alkaline phosphatase-labeled anti-human IgG antibody(manufactured by BIO SOURCE) is added, and incubated at room temperaturefor 1 hour. After washing, the substrate solution is added andincubated, followed by the measurement of absorbance at 405 nm using theMICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate theconcentration of the desired antibody.

FCM Analysis

Reactivity of the antibody of the present invention with lymphocytes maybe examined by flow cytometry (FCM) analysis. As the cells, establishedcell lines or freshly isolated cells can be used. As established celllines, there may be used myeloma-derived RPMI8226 (ATCC CCL 155),myeloma-derived U266 (ATCC TIB 196), myeloma-derived KPMM2,myeloma-derived KPC-32, and plasmacytoma-derived ARH-77 (ATCC CRL 1621),and the like.

After washing the above cells in PBS(−), 100 μl of antibody or a controlantibody diluted to 25 μg/ml in the FACS buffer (PBS(−) containing 2%bovine fetal serum and 0.05% sodium azide) is added thereto, which isthen incubated on ice for 30 minutes. After washing with the FACSbuffer, 100 μl of 25 μg/ml FITC-labeled goat anti-mouse antibody (GAM,manufactured by Becton Dickinson) is added thereto, which is thenincubated on ice for 30 minutes. After washing with the FACS buffer, thecells are suspended in 600 μl of 1 ml of the FACS buffer, and each cellmay be measured for its fluorescence intensity using the FACScan(manufactured by Becton Dickinson).

Screening Method

In order to screen the expression enhancer of HM1.24 antigen, forexample, the cells that have not been stimulated and that are notexpressing HM1.24 antigen or the cells that at least are expressing theantigen are determined using FCM analysis. For example, the cellsdescribed in Examples and a test substance are incubated for 1-2 days,and then stained with mouse anti-human HM1.24 antibody as a primaryantibody. The cells are washed and further stained with FITC-labeledanti-mouse IgG antibody as a secondary antibody. Finally, after washingthe cells, the fluorescence intensity of FITC is measured by a flowcytometer.

Furthermore, instead of the above indirect staining, FCM analysis bydirect staining may be used in which the cells are treated with a highconcentration of immunoglobulin, and then stained, after blocking Fcreceptors, with FITC-labeled anti-human HM1.24 antibody.

It is also possible to screen expression enhancers of HM1.24 antigen bythe reporter gene assay using the HM1.24 promoter sequence. As thereporter gene, luciferase can be used. A plasmid is constructed thatcontains the HM1.24 promoter sequence upstream of the reporter gene,after which it is transformed into the cells, and the cells obtained arecultured with a test substance for 1-2 days, and the cells recovered aresubjected to FCM analysis to screen drugs that enhance the expression ofHM1.24 antigen.

Cytotoxicity

Measurement of ADCC Activity

The antibody for use in the present invention is one which has, forexample, an ADCC activity as the cytotoxicity.

According to the present invention, the ADCC activity on hematopoietictumor cells can be measured in the following manner. First, mononuclearcells (E)are isolated as the effector cells from human peripheral bloodor bone marrow by the gravity centrifuge method.

As the target cells (T), RPMI8226 (ATCC CCL 155), U266 (ATCC TIB 196),KPMM2, KPC-32, ARH-77 (ATCC CRL 1621), HEL, cells derived from patientsor the like is labeled with ⁵¹Cr to be prepared as the target cells.Subsequently, to the labeled target cells is added the antibody to bemeasured for the ADCC activity and incubated. Effector cells at asuitable ratio to the target cells are then added and incubated.

After incubation, the supernatant is removed and measured forradioactivity using a gamma counter. At this time 1% NP-40 can be usedfor measurement of the maximum free radioactivity. The cytotoxicity (%)can be calculated as (A−C)/(B−C)×100, in which A is radioactivity (cpm)liberated in the presence of the antibody, B is radioactivity (cpm)liberated by NP-40, and C is radioactivity (cpm) liberated by the mediumalone containing no antibody.

Enhancement of Cytotoxicity

In order to exhibit cytotoxicity such as an ADCC activity, it ispreferred to use Cγ, in particular Cγ1 and Cγ3 as the constant region (Cregion) of antibody in humans. Furthermore, a more potent ADCC activityor CDC activity can be induced by adding, altering, or modifying part ofthe amino acids in the C region of antibody.

By way of example, there can be mentioned the conversion of IgG to anIgM-like polymer by amino acid substitution (Smith, R. I. F. & Morrison,S. L. BIO/TECHNOLOGY (1994) 12, 683-688), the conversion of IgG to anIgM-like polymer by amino acid addition (Smith, R. I. F. et al., J.Immunology (1995) 154, 2226-2236), the expression of a tandemly-ligatedgene encoding L chain (Shuford, W. et al., Science (1991) 252, 724-727),the dimerization of IgG by amino acid substitution (Caron, P. C. et al.,J. Exp. Med. (1992) 176, 1191-1195, Shopes, B., J. Immunology (1992)148, 2918-2922), the dimerization of IgG by chemical modification(Wolff, E. A. et al., Cancer Res. (1993) 53, 2560-2565), and theintroduction of the effector function by altering an amino acid(s) inthe hinge region of antibody (Norderhaug, L. et al., Eur. J. Immunol.(1991) 21, 2379-2384) and the like.

These can be accomplished by means of the oligomer site-specificmutagenesis using a primer, the addition of a base sequence using arestriction enzyme cleavage site, and the use of a chemical modifierthat creates a covalent bond.

Treatment of Patients

One embodiment of the present invention concerns a method of treatinghematopoietic tumors by administering to the patient a pharmaceuticalagent that enhances the amount expressed of HM1.24 antigen, and apharmaceutical agent that induces the expression of HM1.24 antigen inthe cell that normally is not expressing HM1.24 antigen on the cellsurface and anti-HM1.24 antibody. The hematopoietic tumors are, forexample, myeloma, preferably multiple myeloma, lymphocytic tumors suchas lymphoma, preferably Hodgkin's disease or non-Hodgkin lymphoma,lymphocytic leukemia, preferably acute T lymphocytic leukemia, chronic Tlymphocytic leukemia, acute B lymphocytic leukemia, chronic Blymphocytic leukemia, and myelocytic leukemia for example acutemyelocytic leukemia and chronic myelocytic leukemia. Furthermore, acuteleukemia belonging to L1-L3 and M0-M7 in the FAB Classification is alsoincluded.

A pharmaceutical agent that enhances the amount expressed of HM1.24antigen, and a pharmaceutical agent that induces the expression ofHM1.24 antigen in the cell that normally is not expressing HM1.24antigen on the cell surface and anti-HM1.24 antibody is preferablyinterferon-α, interferon-γ, the IRF-2 protein, or a vector containingDNA encoding the IRF-2 protein, and preferably interferon-α andinterferon-γ. The dosage of interferon-α and interferon-γ is, whenadministered to humans, is an amount that attains the highest bloodlevel of 1-10000 I.U./ml (International Units) , more preferably 5-1000I.U./ml, even more preferably 5-500 I.U./ml, and most preferably 5-50I.U./ml.

In the case of intravenous administration, preferably ten thousand toten million I.U., more preferably one hundred thousand to ten millionI.U., even more preferably fifty thousand to five million I.U., mostpreferably one million to five million I.U. is given per administration.Interferon and anti-HM1.24 antibody may be administered together orseparately. In the latter case, preferably interferon is given first,followed by the administration of anti-HM1.24 antibody within 96 hours.The interval between the interferon administration and the anti-HM1.24antibody administration is not limited as long as the amount expressedof HM1.24 antigen is being enhanced by the administration of interferon,but it is preferably within 96 hours, more preferably 72 hours, stillmore preferably 48 hours.

Alternate administration of interferon and anti-HM1.24 antibody for aplurality of times depending on the clinical response of the patient iswithin the scope of the present invention. The route of administrationis preferably given directly into the blood circulation, and intravenousadministration or intraarterial administration is preferred. Continuedadministration is possible and intravenous drip may be used. Theadministration may also be subcutaneous or intramuscular administration.

Another aspect of the present invention concerns a therapeutic agent ora pharmaceutical composition for the treatment of hematopoietic tumorscomprising interferon-α or interferon-γ and anti-HM1.24 antibody. Thetherapeutic agent of the present invention may contain apharmaceutically acceptable vehicle that has been used for interferonand antibody preparations, such as physiological saline or 5% dextran,together with a common stabilizer or a excipient.

Another aspect of the present invention provides a kit for treating apatient with hematopoietic tumors, comprising a pharmaceuticalcomposition comprising anti-HM1.24 antibody as an active ingredient andan instruction manual that contains description of combined therapy withinterferon-α or interferon-γ.

Another aspect of the present invention provides a pharmaceuticalcomposition comprising anti-HM1.24 antibody as an active ingredient fortreating a patient with hematopoietic tumors, wherein said compositionis used in combination with interferon-α or interferon-γ.

Another aspect of the present invention relates to the administration ofa gene encoding a substance that enhances or induces the expression ofHM1.24 antigen. For example, the DNA sequences of interferon-α,interferon-γ, IRF-2 are known and can be administered to patients byintegration into the desired vector. As the vector, they are insertedinto adenovirus vector (for example pAdexLew) or retrovirus vector (forexample pzIPneo), which are then administered to the living body. Themethod of administration may be ex vivo or in vivo. Alternatively, nakedDNA may be administered.

EXAMPLES Example 1 Enhancement of the Amount of HM1.24 Antigen Expressedin Myeloma Cells by Interferon-α

A human myeloma cell line U266 (ATCC TIB 196) and myeloma cells derivedfrom the bone marrow of a patient with multiple myeloma were cultured ina RPMI1640 medium (Sigma, St Louis, Mo., USA) containing 10% fetalbovine serum (Whittaker Bioproducts, Inc., Walkersville, Md., USA) in a5% carbon dioxide incubator at 37° C. The hybridoma that produces mouseanti-HM1.24 antibody has been internationally deposited as FERM BP-5233(deposition date: Apr. 27, 1995) with the National Institute ofBioscience and Human Technology, of 1-3, Higashi 1-chome, Tsukuba city,Ibaraki Pref.

Myeloma cells (1×10⁵/ml) were cultured in the presence or absence of1000 U/ml of the natural type interferon-α (otsuka Pharmaceutical,Tokyo) for 48 hours, and changes in HM1.24 antigen (the base sequenceencoding this is shown in SEQ ID NO: 1) were determined by flowcytometry. After the cells were washed with phosphate buffer (Gibco BRL,Grand Island, N.Y., USA) supplemented with 0.1% bovine serum albumin(Sigma, St Louis, Mo., USA) and 0.02% sodium azide, they were suspendedinto PBS (100 μl) supplemented with human immunoglobulin (3 mg/ml, GreenCross, Osaka), and were allowed to react at 4° C. for 15 minutes.

Thereafter, 2 μl of FITC-human IgG1 (1 mg/ml) or FITC-anti-HM1.24antibody (1 mg/ml) was added to stain at 4° C. for 60 minutes. When thepatient's myeloma cells were used, 20 μl of PE-anti-CD38 (BectonDickinson, San Jose, Calif., USA) was added for double staining toidentify the myeloma cells. After staining, the cells were washed twicewith PBC, and were stored in PBS containing 1% paraformaldehyde (WakoPure Chemical Industries, Ltd., Osaka). Subsequently, the expression ofHM1.24 antigen was analyzed using a flow cytometer (EPICS XL, Coulter,Hialeah, Fla., USA).

As a result, a myeloma cell line U266 (FIG. 1) and the patient's myelomacells (FIG. 2) were expressing HM1.24 antigen at a condition of nostimulation, and the stimulation with interferon-α further increased theamount expressed of HM1.24 antigen.

Interferon-α further enhanced the expression of HM1.24 antigen in themyeloma cell, and increased the number of anti-HM1.24 antibodies thatbind to the myeloma cell. Since the therapeutic anti-tumor effect byanti-HM1.24 antibody is proportional to the number of antibodies thatbound, treatment with anti-HM1.24 antibody after the administration ofinterferon-α is expected to provide a therapy that enhances thetherapeutic effect by antibody and further enhances effectiveness.

Example 2 Analysis of the Expression Function of HM1.24 Antigen by theReporter Gene Analysis

In order to investigate whether the expression induction of antigen isregulated by the HM1.24 promoter region, the reporter gene at thepromoter region was analyzed.

The gene (SEQ ID NO: 3) of the HM1.24 promoter region was obtained byPCR cloning. Genomic DNA was prepared from human peripheral bloodmononuclear cells using the DNAzol reagent (GIBCO). With the genomic DNAobtained as the template, using primer HM2k(aaaggtaccagctgtctttctgtctgtcc) (SEQ ID NO: 4) and BST2B(atagtcatacgaagtagatgccatccag) (SEQ ID NO: 5), PCR (94° C. for 1 min,55° C. for 1 min, 72° C. for 1 min, 30 cycles) was performed usingTaKaRa Taq (Takara Shuzo, Ohtsu) in the Thermal Cycler 480(Perkin-Elmer, Calif., USA).

An about 2 kb fragment obtained was treated with restriction enzymesKpnI and BglII (Takara Shuzo), and was cloned into the KpnI-BglII siteof a reporter gene plasmid pGL3-basic (Promega, Wis., USA) using the DNAligation kit ver. II (Takara Shuzo) to transform into competent E. coliJM109 (Nippongene). The transformed E. coli was cultured at 37° C. inthe LB medium containing 100 μg/ml ampicillin, and the plasmid wasprepared using the QIAGEN plasmid maxi kit (QIAGEN, Hilden, Germany).

The plasmid HM-2k/GL3 obtained was treated with restriction enzymes KpnIand XhoI, from which a deletion clone was constructed using the deletionkit (Takara Shuzo) for kilo-sequence to obtain a plasmid HM-493/GL3containing from the transcription initiation point to −493 bp upstream.Furthermore, HM-2k/GL3 was treated with restriction enzymes KpnI andAf1II, from which a deletion clone was constructed as described above,and HM-151/GL3 and HM-77/GL3 containing from the transcriptioninitiation point to −151 bp or −77 bp upstream were obtained.

For the introduction of the plasmid into the cell, thepolyethyleneimine-Transferrinfection Kit (Tf PEI-Kit) (BenderMedSystems, Vienna, Austria) was used, and for the luciferase assay theDual-Luciferase Reporter Assay System (Promega) was used. The cell linewas cultured overnight in RPMI-1640 containing 50 μm Defferrioxamine and10% FBS. In order to form a complex of the plasmid to be introduced withTf-PEI, a mixture of the reporter gene plasmid at a final concentrationof 20 μg/ml, 0.4 μg/ml of pRL-SV40, and 1 μg/ml of Tf-PEI reagent wasprepared and was incubated at room temperature for 20 minutes. 5×10⁵cells/ml of cells were added at three volumes of the Tf-PEI/plasmidmixture, and was incubated at 37° C. for four hours, washed with themedium, and 100 μl per well at a concentration of 2×10⁵ cells/ml wascultured in a 96-well flat-bottomed plate.

IFN-α was added to a final concentration of 0, 10, 100, or 1000 U/ml,which was cultured at 37° C. for two days. After the cells were washedin PBS(−), they were dissolved in 20 μl of the Passive Lysis Buffer, sixμl of which was applied to the C96 White Polysorp Fluoronunc plate(Nunc). Using the Luminoskan (Labsystems), luminescence intensity wasmeasured for Firefly and Renila at 30 μl of the substrate solution and ameasurement time of 10 seconds. The measured values were corrected byFirefly/Renila, and the relative activity was determined with thecontrol (medium) as one.

As a result, the luciferase activity of the reporter was increased in aIFN-α concentration dependent manner for both of the upstream 2 kbp and493 bp, confirming that the enhanced transcription activity of thepromoter region causes the expression induction of the antigen (FIG. 3).

Furthermore, the result of an experiment in which the reporter plasmid151 bp or 77 bp upstream of the transcription initiation point was used,an enhanced luciferase activity by IFN-α stimulation was observed forthe reporter plasmid 151 bp upstream. On the other hand, no changes inactivity were noted by IFN-α stimulation in the reporter plasmid 77 bpupstream (FIG. 4). In the region of 77-151 bp, a sequence having a highhomology with GAS element and ISRE was present, and since it is atranscription regulatory factor that is activated in response to IFN-αstimulation, the transcription regulatory factor of the IRF family wasshown to be involved in the activity.

Example 3 Enhancement of the Amount Expressed of HM1.24 Antigen inMyeloma Cells by Interferon-γ

According to the method described in Example 1, 1000 U/ml of the naturaltype interferon-γ (R & D System) was used for analysis. As a result,increases in the amount expressed of HM1.24 antigen were observed inboth of the myeloma cell line U266 (FIG. 5) and the patient's myelomacells (FIG. 6) as for interferon-α.

Example 4 Binding of IRF-2 to the HM1.24 Promoter Region

In order to identify the transcription factor that binds to the HM1.24promoter region, the Electrophoresis Mobility Shift Assay (EMSA) withthe HM1.24 promoter region as the probe was performed as follows toidentify IRF-2 as the binding factor.

(1) Preparation of Nuclear Extract

The myeloma cells U266-B1 (ATCC-TIB196) were cultured in the RPMI-1640medium (GIBCO-BRL) containing 10% FBS (HyClone) at 37° C. in a 5% CO₂incubator. In order to stimulate the cells by interferon-α (IFN-α)(Pepro Tech EC), IFN-α was added to the medium to a final concentrationof 1000 U/ml, and the cells were recovered at 30 minutes, two hours,four hours, and eight hours after the addition. The cells were suspendedinto cold PBS (−), centrifuged at 1,000 rpm to discard the supernatant,and suspended in a 10 mM Tris, 10 mM NaCl, and 6 mM MgCl₂ solution.

After allowing to stand in ice for five minutes, centrifugation wasrepeated, and the supernatant was discarded. The cells were suspendedinto 10 mM Tris, 10 mM NaCl, 6 mM MgCl₂, 1 mM DTT, 0.4 mM PMSF, 1 mMNa₃VO₄. The cells were homogenized on ice using a glass homogenizer,centrifuged at 6000 g for three minutes, and the supernatant wasdiscarded. The cells were suspended into the extraction buffer (20%glycerol, 20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl_(2,) 0.2 mM EDTA, 0.2 mMPMSF, 1 mM DTT, 0.1 mM Na₃VO₄, 2 mg/ml aprotinin, and 5 mg/mlleupeptin), and then was allowed to stand in ice for 20 minutes. It wascentrifuged at 12000 g for 10 minutes, and the supernatant wasrecovered.

(2) Preparation of the Labeled Probe

As the probe, ISRE2 was constructed that contains sequences (ttcccagaaa(SEQ ID NO: 10) and ggaaactgaaact (SEQ ID NO: 11)) having a homologywith GAS (IFN-γ activation site: the GAS consensus sequence is ttncnnnaa(SEQ ID NO: 8)) and ISRE (IFN-α stimulation response factor: the ISREconsensus sequence is ngaaanngaaact (SEQ ID NO: 9)), at the HM1.24promoter region. Thus, oligo DNA ISRE-F2 (aatttctgggaaactgaaactgaaaacct(SEQ ID NO: 12)) and ISRE-R2 (aattaggttttcagtttcagtttcccaga (SEQ ID NO:13)) were mixed and annealed to form a double stranded DNA probe ISRE2.

Furthermore, oligo DNA adp-1 (catggcatctacttcgtatgactattgcagagtgcc (SEQID NO: 14)) and adp-2 (catgggcactctgcaatagtcatacgaagtagatgc (SEQ ID NO:15)) were mixed and annealed to form an unrelated probe adp. Probes werelabeled using the Band Shift Kit (Amersham Pharmacia Biotech) accordingto the standard protocol. Thus, 50 ng of double stranded DNA constructedas above was subjected to the polymerase reaction of the Klenow fragmentin a reaction solution containing [α-³²P]dATP (20 μCi) (AmershamPharmacia Biotech) at 37° C. for one hour. The solution at the end ofthe reaction was diluted two-fold and then was loaded to the Nick SpinColumn (Amersham Pharmacia Biotech), and after centrifugation at 1600rpm for four minutes, the solution was recovered to prepare a labeledprobe.

(3) Changes with Time in the Binding Factor Produced by Stimulation withIFN-α

According to the standard protocol of the Band Shift Kit (AmershamPharmacia Biotech, N.J., USA), the following procedure was performed. To5 μg of the extracts prepared as the time elapse in the above (1), wereadded 2 μl of the 10× biding buffer (100 mM Tris-HCl, pH 7.5, 500 mMNaCl, 5 mM DTT), 4 μl of 50% glycerol, 1 μl of 1% NP-40, and 1 μl ofpoly(dI-dC)·poly(dI-dC), and then 2 μl of the ³²p labeled ISRE-2 probeprepared in the above (2), to which water was added to a total volume of20 μl, was added and this reaction mixture was incubated at roomtemperature for 20 minutes to allow for the binding of the possiblebinding factors that may be present in the above extract and said ³²Plabeled ISRE-2 probe.

To 18 μl of the reaction mixture was added 2 μl of the 10× stainsolution (attached to the kit), which was electrophoresed in 1×Tris-glycine buffer (25 mM Tris, 190 mM glycine, 1 mM EDTA, pH 8.1) on a7.5% acrylamide gel, and then, after electrophoresis, the gel wasattached to a filter paper to transfer protein to the filter paper. Thefilter paper dried with a gel drier was exposed to X-ray film to detectsignals.

For comparison, a reaction solution [(NEC−)] to which no extract wasadded, a reaction solution [0 h] to which an extract from the cellculture that was cultured without stimulation by interferon-α was added,a reaction solution {8 h (+cold)] in which 50 ng of a nonlabeled ISRE2probe was added in stead of the labeled probe to the extract of 8hour-culture, and a reaction solution [8 h (+cold unrelated)] in which50 ng of an unrelated probe adp was added to the extract of 8hour-culture were prepared, and were processed as described above todetect signals.

The result is shown in FIG. 7. As can be seen from this figure, asubstance that binds to a double stranded DNA corresponding to part ofthe HM1.24 promoter increased with time in the U266-B1 cells culturedunder the stimulation by interferon.

(4) The Identification of a Transcription Factor by Reaction withVarious Antibodies

In a manner similar to that described in the above (1), the myelomacells U266-B1 (ATCC-TIB196) were cultured for eight hours in thepresence of 1000 U/ml of interferon-α to prepare an extract. Thefollowing procedure was performed according to the standard protocol ofthe Band Shift Kit (Amersham Pharmacia Biotech). Thus, 2 μg of antibodywas added to 5 μg of the extract, and incubated at room temperature for15 minutes to obtain an extract/antibody reaction solution. To 2 μl ofthe 10× binding buffer attached to the kit, 4 μl of 50% glycerol, 1 μlof 1% NP-40, and 1 μl of Poly(dI-dC)·Poly(dI-dC) were added 2 μl of theabove extract/antibody reaction solution and 2 μl of the labeled probeprepared in the above (2), to which water was added to make the totalvolume 20 μl, and the reaction mixture was incubated at room temperaturefor 20 minutes.

The reaction mixture was subjected to electrophoresis as described inthe above (3) to detect signals.

As the above antibody, the following antibodies (all are from Santa CruzBiotechnology) were used:

Anti-human STAT1 p84/p91 (E-23): (description) rabbit polyclonalantibody (SC-346X)

Anti-human STAT2 (C-20): rabbit polyclonal antibody (SC-476X)

Anti-mouse STAT3 (K-15): rabbit polyclonal antibody (SC-483X)

Anti-human ISGF-3γ p48 (C-20): rabbit polyclonal antibody (SC-496X)

Anti-human IRF-1 (C-20): rabbit polyclonal antibody (SC-497X)

Anti-human IRF-2 (C-19): rabbit polyclonal antibody (SC-498X)

Anti-mouse ICSAT (M-17): rabbit polyclonal antibody (SC-6059X)

As a control, a reaction solution that uses an extract of the cellscultured without interferon stimulation [0 h]; a reaction solution inwhich an extract of the cells cultured for eight hours under thestimulation with 1000 U/ml interferon-α was added, and no antibody wasadded [8 h]; a reaction solution in which 50 ng of the nonlabeled ISRE2probe was added instead of the labeled ISRE2 [8 h (+cold)]; and areaction solution in which 50 ng of the nonlabeled dp probe was added instead of the labeled ISRE2 probe [8 h (+unrelated cold)] were preparedand processed as described above.

The result is shown in FIG. 8. As can be seen from this figure, it wasshown that the component that binds to the labeled ISRE2 probe in theextract from the cells cultured under the stimulation with interferon-αbinds only to anti-IRF-2 antibody, and the factor that binds to andthereby activates the HM1.24 promoter is a transcription factor IRF-2.

Example 5 Confirmation of the HM1.24 Promoter Activation with IRF-2

Effect on HM1.24 promoter activity by IRF-2 co-expression was determinedby the reporter gene assay using the U266 cells, and it was revealedthat IRF-2 actually has the transcription activation activity of theHM1.24 promoter. In the following experiment, a myeloma cell lineU266-B1 (ATCC TIB196) was used. The cells were cultured in the RPMI-1640medium (GIBCO) (referred to hereinafter as the medium) containing 10%FBS (GIBCO BRL) in an 5% CO₂ incubator.

(1) Construction of a Plasmid Containing the HM1.24 Promoter Region

The gene of the HM1.24 promoter region was obtained by PCR cloning. Fromhuman peripheral blood mononuclear cells, genomic DNA was prepared usingthe DNAzol reagent (GIBCO). With the genomic DNA obtained as thetemplate, using primer HM2k (aaaggtaccagctgtctttctgtctgtcc) (SEQ ID NO:16) and BST2B (atagtcatacgaagtagatgccatccag) (SEQ ID NO: 17), PCR (94°C. for 1 min, 55° C. for 1 min, 72° C. for 1 min, 30 cycles) wasperformed using TaKaRa Taq (Takara Shuzo, Ohtsu) in the Thermal Cycler480 (Perkin-Elmer, Calif., USA).

An obtained about 2 kb fragment was treated with restriction enzymesKpnI and BglII (Takara Shuzo), and was cloned into the KpnI-BglII siteof a reporter gene plasmid pGL3-basic (Promega, Wis., USA) using the DNAligation kit ver. II (Takara Shuzo) to transform competent E. coli JM109(Nippongene). The transformed E. coli was cultured at 37° C. in the LBmedium containing 100 μg/ml ampicillin, and a plasmid was prepared usingthe QIAGEN plasmid maxi kit (QIAGEN, Hilden, Germany).

The plasmid HM-2k/GL3 obtained was treated with restriction enzymes KpnIand XhoI, from which a deletion clone was constructed using the deletionkit (Takara Shuzo) for kilo-sequence to obtain a plasmid HM-491/GL3containing up to −491 bp upstream of the transcription initiation point.Furthermore, HM-2k/GL3 was treated with restriction enzymes KpnI andAflII, from which a deletion clone was constructed as described above,and HM-151/GL3 containing −151 bp upstream of the transcriptioninitiation point was obtained.

Furthermore, with HM-2k/GL3 as the template, using primer 10S(tttcggtacctaattaatcctctgcctg) (SEQ ID NO: 18) and GL primer 2(ctttatgtttttggcgtcttcca) (SEQ ID NO: 19), PCR (94° C. for 1 min, 55° C.for 1 min, 72° C. for 1 min, 30 cycles) was performed using TaKaRa Taq(Takara Shuzo, Ohtsu) in the Thermal Cycler 480 (Perkin-Elmer, Calif.,USA). The fragment obtained was treated with restriction enzymes KpnIand BglII (Takara Shuzo), and was cloned into the KpnI-BglII site of areporter gene plasmid pGL3-basic (Promega, Wis., USA) using the ligationhigh (Toyobo) to transform competent E. coli JM109 (Nippongene).

The transformed E. coli was cultured at 37° C. in the LB mediumcontaining 100 μg/ml ampicillin, and a plasmid was prepared using theQIAGEN plasmid maxi kit (QIAGEN, Hilden, Germany). A plasmid HM-125/GL3was thus obtained that contains up to 125 bp upstream of thetranscription initiation point. Furthermore, with HM-2k/GL3 as thetemplate, using primer HMP700 (aaaggtaccagagtttacctggtatcctgg) (SEQ IDNO: 20) and GL primer 2, PCR was performed in a similar procedure, andby introducing the fragment into the KphI-BglII site of pGL3-basic,HM-700/GL3 containing up to about 700 bp upstream of the transcriptioninitiation point was obtained.

Furthermore, with HM-2k/GL3 as the template, using primer HMP700 and11A′ (cagaggattaattaggtaccgaaagagaggtgggctttt) (SEQ ID NO: 21), PCR (98°C. for 15 seconds, 65° C. for 2 seconds, 74° C. for 30 seconds, 25cycles) was performed using the KOD polymerase (Toyobo) in the ThermalCycler 480 (Perkin-Elmer, Calif., USA). The fragment obtained wasinserted into the pCR4 Blunt-TOPO vector using the Zero Blunt TOPO PCRcloning kit for sequencing ver. B (Invitrogen). The plasmid obtained wastreated with a restriction enzyme KpnI, and an about 550 bp fragment wasrecovered, which was introduced into the KpnI site of HM-125/GL3 usingthe ligation high. Thus, dSIRE/GL3 lacking −25 to about −145 upstream ofthe transcription initiation point was obtained.

(2) Construction of IRF-2 Expression Plasmid

The IRF-2 expression plasmid was constructed as follows. From the U266cells for which eight hours have elapsed after stimulation withinterferon-α (1000 U/ml), total RNA was extracted using the TRIzolreagent (GIBCO BRL). With RNA obtained by using the First-strand cDNASynthesis kit (Pharmacia) as the template, using NotI-d(T)₁₈ as theprimer, a reverse transcription reaction was performed at 37° C. for onehour. With the cDNA obtained as the template, using IRF2-F2(ttgtattggtagcgtgaaaaaagc) (SEQ ID NO: 22) and IRF2-R2(cagctagttcacattatctcgtcc) (SEQ ID NO: 23) as primers, PCR (94° C. for45 seconds, 60° C. for 45 seconds, 72° C. for 60 seconds, 40 cycles) wasperformed using LA-Taq (Takara Shuzo).

With the PCR reaction as the template, using IRF2-F1(agagggtaccatgccggtggaaaggatgcg) (SEQ ID NO: 24) and IRF2-R1(agtcggtaccttaactgctcttgacgcggg) (SEQ ID NO: 25) as primers, PCR (94° C.for 45 seconds, 60° C. for 45 seconds, 72° C. for 60 seconds, 30 cycles)was performed using the KOD polymerase (Toyobo). The fragment obtainedwas treated with a restriction enzyme KpnI, and then introduced into theKpnI site of an expression plasmid pTracer-CMV (Invitrogen) using theligation high (Toyobo) to obtain a IRF-2 expression plasmidpIRF-2/Tracer.

(3) Measurement of the Reporter Gene Activity

For the introduction of the plasmid into the cells, thepolyethyleneimine-Transferrinfection Kit (Tf PEI-Kit) (BenderMedSystems, Vienna, Austria) was used, and for the luciferase assay theDual-Luciferase Reporter Assay System (Promega) was used. The cell linewas cultured overnight in RPMI-1640 containing 50 μM Defferrioxamine and10% FBS. In order to form a complex of the plasmid to be introduced withTf-PEI, a mixture of the reporter gene plasmid at a final concentrationof 20 μg/ml, 20 μg/ml of pIRF-2/Tracer or pTracer-CMV, 0.4 μg/ml ofpRL-SV40, and 1 μg/ml of Tf-PEI reagent was prepared, and was incubatedat room temperature for 20 minutes.

5×10⁵ cells/ml of cells were added at three times the volume of theTf-PEI/plasmid mixture, and was incubated at 37° C. for four hours,washed with the medium, and 100 μl per well at a concentration of 2×10⁵cells/ml was cultured in a 96-well flat-bottomed plate. IFN-α was addedto a final concentration of 0 or 1000 U/ml, which was cultured at 37° C.for two days. After the cells were washed in PBS(−), the cells weredissolved in 20 μl of the Passive Lysis Buffer, six μl of which wasapplied to the C96 White Polysorp Fluoronunc plate (Nunc). Using theLuminoskan (Labsystems), luminescence intensity was measured for Fireflyand Renila at 30 μl of the substrate at a measurement time of 10seconds. The measured values were corrected by Firefly/Renila to obtainthe correct relative activity.

(4) Result

The HM1.24 promoter reporter plasmid and the IRF-2 expression plasmidwere introduced into the U266 cells, and the reporter activity wasdetermined (FIG. 9). As a result, the luciferase activity was increasedin −700 and −151 containing the ISRE motif sequence that is a IRF-2binding site by IRF-2 co-expression. On the other hand, no changes inluciferase activity were noted in dISRE/GL3 that lacks the ISRE sequenceby IRF-2 co-expression. This result indicated that IRF-2 binds to theISRE region of the HM1.24 promoter and enhances its transcriptionactivity.

(5) Confirmation of Enhanced Expression of HM1.24 Antigen by OverExpression of IRF-2

For changes in the amount expressed of HM1.24 antigen by IRF-2, theIRF-2 expression plasmid (PIRF-2/Tracer) or the control plasmid(pTracer/CMV) is introduced into the U266 cells in the method describedabove, then cultured for 1-2 days, from which the cells are recovered,and then stained with mouse anti-human HM1.24 antibody as a primaryantibody. The cells are washed, and further stained with FITC-labeledanti-mouse IgG antibody as a secondary antibody. After washing thecells, the FITC fluorescence intensity of the cells was measured by aflow cytometer. It is confirmed that in the cells in which the IRF-2expression plasmid was introduced there are more cells having a highFITC intensity compared to the cells in which the control plasmid wasintroduced.

Example 6 Enhancement of the Amount Expressed of HM1.24 Antigen byInterferon-α in Lymphatic Tumor Cells and Expression Induction of HM1.24Antigen in Myelocvtic Leukemia Cells

A human myelocytic leukemia cell line HEL (Japanese Cancer ResearchResources, Tokyo, Japan) and tumor cells derived from the bone marrowand lymph node of patients with hematopoietic tumors were cultured inthe method described in Example 1. Interferon-α used is the same as inExample 1, and the method of stimulation by interferon-α and the methodof flow cytometry were performed in a similar manner to that describedin Example 1. As the FITC-anti-HM1.24 antibody, FITC-humanizedanti-HM1.24 antibody (1 mg/ml) was used (see International PatentPublication WO 98/14580, the versions of the light chain and the heavychain variable region are RVLa and RVHs, respectively). Twenty μl ofPE-anti-CD33 antibody for the identification of myelocytic leukemiacells, PE-anti CD19 antibody for the identification of B lymphocyticlymphoma, and PE-anti CD4 antibody (all are from Pharmingen, San Diego,Calif., USA) for the identification of T lymphocytic lymphoma wereadded, and double-stained (FIGS. 10 to 12).

As a result, under no stimulation with IFN-α, very little or littleexpression of HM1.24 antigen on the cell surface of HEL and tumor cellsderived from patients with acute myelocytic leukemia was observed,indicating that the expression of HM1.24 antigen under no stimulationwith IFN-α is substantially not observed in these tumor cells. On theother hand, in tumor cells derived from patients with acute lymphocyticleukemia, B lymphocytic non-Hodgkin lymphoma, and T lymphocyticnon-Hodgkin lymphoma, the expression of HM1.24 antigen on the cellsurface was observed even under no stimulation with IFN-α. In contrast,when stimulated with IFN-α, the expression of HM1.24 antigen was inducedin HEL and tumor cells derived from patients with acute myelocyticleukemia.

In these cells that substantially do not express HM1.24 antigen, IFN-αcould cause the expression of HM1.24 antigen. Also, in tumor cellsderived from patients with acute lymphocytic leukemia, B lymphocyticnon-Hodgkin lymphoma, and T lymphocytic non-Hodgkin lymphoma, the amountexpressed of HM1.24 antigen increased. In these five types of tumorcells, the amount of HM1.24 antigen expressed by IFN-α stimulation wasalmost the same. These results indicate that IFN-α exhibits the effectof inducing or enhancing the expression of HM1.24 antigen forhematopoietic tumor cells in general.

Example 7 Enhancement of ADCC Activity of Anti-HM1.24 Antibody byInterferon α

A human myelocytic leukemia cell line HEL was used as the target cell.To HEL (1×10⁶ cells), 0.1 mCi of 51 Cr-sodium chromate (New EnglandNuclear, Boston, Mass., USA) was added and was allowed to stand at 37°C. for 1 hour. Then the cells were washed three times in RPMI1640, and1×10⁴ cells were dispensed into a round-bottomed 96-well plate(Corning).

After adding various concentrations of humanized anti-HM1.24 antibody(International Patent Publication WO 98/14580, the versions of the lightchain and the heavy chain variable region are RVLa and RVHs,respectively), peripheral blood mononuclear cells (5×10⁵) from normalhealthy subjects were added, and were allowed to stand at 37° C. for 4hours. The radioactivity of the culture supernatant was counted by agamma counter, and cytotoxicity by ADCC was calculated by the followingequation. The maximum value was measured by disrupting the cells byadding 1% NP40 to the target cells. The minimum value was measured byadding the culture liquid RPMI1640 alone (FIG. 13).ADCC activity (%)=(measured value−minimum value)/(maximum value−minimumvalue)

As a result, in the absence of stimulation by IFN-α, no concentrationsof humanized anti-HM1.24 antibody caused marked ADCC activity. Whenstimulated by IFN-α, marked ADCC activity due to humanized anti-HM1.24antibody was noted. Furthermore, ADCC activity was dependent on antibodyconcentration, which indicated that the expression of HM1.24 antigen onthe cell surface induced ADCC activity.

The result indicates that the enhanced amount expressed of HM1.24antigen on the surface of tumor cells due to stimulation by IFN-α etc.leads to enhancement in cytotoxicity due to anti-HM1.24 antibody, i.e.enhancement in anti-tumor effect and, therefore, that the combined useof an expression-enhancing agent such as IFN-α of anti-HM1.24 antibodycould attain an equivalent effect with the use of a smaller amount ofanti-HM1.24 antibody. It also indicates that, even in hematopoietictumor cells for which a marked effect cannot be expected withanti-HM1.24 antibody alone, the combined use of an expression-enhancingagent such as IFN-α and anti-HM1.24 antibody is likely to attain ananti-tumor effect.

1. An expression-enhancing or expression-inducing agent of a protein containing the amino acid sequence set forth in SEQ ID NO: 2 in hematopoietic tumor cells, said agent comprising interferon α, interferon γ, or the IRF-2 protein as an active ingredient.
 2. The expression-enhancing or expression-inducing agent according to claim 1 wherein said hematopoietic tumor is leukemia, lymphoma, or myeloma.
 3. The expression-enhancing or expression-inducing agent according to claim 2 wherein said leukemia is acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia.
 4. The expression-enhancing or expression-inducing agent according to claim 2 wherein said lymphoma is Hodgkin's disease, T lymphocytic non-Hodgkin lymphoma, or B lymphocytic non-Hodgkin lymphoma.
 5. The expression-enhancing or expression-inducing agent according to claim 2 wherein said myeloma is multiple myeloma.
 6. A therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors comprising, as an active ingredient, interferon α, interferon γ, or the IRF-2 protein, and an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO:
 2. 7. A therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors comprising, as an active ingredient, an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO: 2, that is capable of use in combination with interferon α, interferon γ, or the IRF-2 protein.
 8. A therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors comprising, as an active ingredient, interferon α, interferon γ, or the IRF-2 protein, that is capable of use in combination with an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID) NO:
 2. 9. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to anyone of claims 6 to 8 wherein said hematopoietic tumor is leukemia, lymphoma, or myeloma.
 10. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to claim 9 wherein said leukemia is acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia.
 11. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to claim 9 wherein said lymphoma is Hodgkin's disease, T lymphocytic non-Hodgkin lymphoma, or B lymphocytic non-Hodgkin lymphoma.
 12. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to claim 9 wherein said myeloma is multiple myeloma.
 13. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to anyone of claims 6 to 8 wherein said antibody is an antibody having cytotoxicity.
 14. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to claim 13 wherein said cytotoxicity is ADCC activity.
 15. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to anyone of claims 6 to 8 wherein said antibody is a monoclonal antibody.
 16. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to claim 15 wherein said antibody is a chimeric antibody, a humanized antibody, or a human antibody.
 17. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to claim 15 wherein said antibody is produced by a hybridoma having the Deposit No. FERM BP-5233.
 18. The therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors according to claim 16 wherein said chimeric antibody or humanized antibody is a chimeric antibody or humanized antibody of an anti-HM1.24 antibody produced by a hybridoma having the Deposit No. FERM BP-5233.
 19. A kit for the treatment of a patient having a hematopoietic tumor, said kit comprising: an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO: 2; and an instruction manual instructing the administration of the above antibody to the patient in combination with an expression-enhancing agent of a protein having the amino acid sequence set forth in SEQ ID NO:
 2. 20. A kit for the treatment of a patient having a hematopoietic tumor, said kit comprising: an expression-enhancing agent of a protein containing the amino acid sequence set forth in SEQ ID NO: 2; an instruction manual instructing the administration of the above antibody agent to the patient in combination with an antibody that specifically binds to a protein having the amino acid sequence set forth in SEQ ID NO:
 2. 21. A kit for the treatment of a patient having a hematopoietic tumor, said kit comprising: an expression-enhancing agent of the amino acid sequence set forth in SEQ ID NO: 2; an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO: 2; and an instruction manual instructing the combined administration of the above agent and the above antibody to the patient.
 22. A method of screening an expression-enhancing agent of an HMI.24 antigen, comprising the steps of: preparing cells having a reporter gene that contains a region of an HM1.24 gene promoter; contacting said cells with a test substance; and detecting expression of the reporter gene and selecting said expression-enhancing agent.
 23. An expression-enhancing agent of the HM1.24 antigen selected by the method according to claim
 22. 24. A pharmaceutical composition for the treatment of hematopoietic tumors wherein said composition comprises the expression-enhancing agent according to claim 23 wherein said composition is capable of being used in combination with an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO:
 2. 25. A method of screening an expression-enhancing agent of the IRF-2 protein comprising the steps of: contacting cells with a test substance; and determining an amount of an IL-2 protein expressed in said cells and selecting said expression-enhancing agent.
 26. An expression-enhancing agent for the IRF-2 protein, selected by the method according to claim
 25. 27. A pharmaceutical composition for the treatment of hematopoietic tumors wherein said composition comprises the expression-enhancing agent according to claim 26 and an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO:
 2. 28. A method for the production of the an expression enhancing or expression-inducing agent for a protein in a hematopoietic tumor cell wherein said protein contains the amino acid sequence set forth in SEQ ID NO:
 2. 29. The method according to claim 28 wherein said hematopoietic tumor cell is leukemia, lymphoma, or myeloma.
 30. The method according to claim 29 wherein said leukemia is acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia.
 31. The method according to claim 29 wherein said lymphoma is Hodgkin's disease, T lymphocytic non-Hodgkin lymphoma, or B lymphocytic non-Hodgkin lymphoma.
 32. The method according to claim 29 wherein said myeloma is multiple myeloma.
 33. A method for the production of a therapeutic agent or a pharmaceutical composition for the treatment of hematopoietic tumors comprising administering an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO:
 2. 34. A method for the production of a therapeutic agent or pharmaceutical composition for the treatment of a hematopoietic tumor comprising administering an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO: 2, which is capable of being used in combination with interferon α, interferon γ, or the IRF-2 protein.
 35. A method for the production of a therapeutic agent or pharmaceutical composition for the treatment of a hematopoietic tumor comprising administering interferon α, interferon γ, or the IRF-2 protein in combination with an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO:
 2. 36. The method according to anyone of claims 33 to 35 wherein said hematopoietic tumor is leukemia, lymphoma, or myeloma.
 37. The method according to claim 36 wherein said leukemia is acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia.
 38. The method according to claim 36 wherein said lymphoma is Hodgkin's disease, T lymphocytic non-Hodgkin lymphoma, or B lymphocytic non-Hodgkin lymphoma.
 39. The method according to claim 36 wherein said myeloma is multiple myeloma.
 40. The method according to anyone of claims 33 to 35 wherein said antibody is an antibody having cytotoxicity.
 41. The method according to claim 40 wherein said cytotoxicity is ADCC activity.
 42. The method according to anyone of claims 33 to 35 wherein said antibody is a monoclonal antibody.
 43. The method according to claim 42 wherein said antibody is a chimeric antibody, a humanized antibody, or a human antibody.
 44. The method according to claim 42 wherein said antibody is an anti-HM1.24 antibody produced by a hybridoma having the Deposit No. FERM BP-5233.
 45. The method according to claim 43 wherein said chimeric antibody or humanized antibody is a chimeric antibody or humanized antibody of an anti-HM1.24 antibody produced by a hybridoma having the Deposit No. FERM BP-5233.
 46. A method for the production of a pharmaceutical composition for the treatment of a hematopoietic tumor, which can be used in combination with an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO: 2 comprising administering the expression-enhancing agent according to claim
 23. 47. A method for the production of a pharmaceutical composition for the treatment of a hematopoietic tumor, which can be used in combination with an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO: 2 comprising administering the expression-enhancing agent according to claim
 26. 48. A method of enhancing or inducing the expression of a protein containing the amino acid sequence set forth in SEQ ID NO: 2 which method comprises administering interferon α, interferon γ, or the IRF-2 protein.
 49. The method according to claim 48 wherein said hematopoietic tumor is leukemia, lymphoma, or myeloma.
 50. The method according to claim 49 wherein said leukemia is acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia.
 51. The method according to claim 49 wherein said lymphoma is Hodgkin's disease, T lymphocytic non-Hodgkin lymphoma, or B lymphocytic non-Hodgkin lymphoma.
 52. The method according to claim 49 wherein said myeloma is multiple myeloma.
 53. A method of treating a hematopoietic tumor which comprises administering interferon α, interferon γ, or the IRF-2 protein, and an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO:
 2. 54. The method according to claim 53 wherein said hematopoietic tumor is leukemia, lymphoma, or myeloma.
 55. The method according to claim 53 wherein said leukemia is acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia.
 56. The method according to claim 53 wherein said lymphoma is Hodgkin's disease, T lymphocytic non-Hodgkin lymphoma, or B lymphocytic non-Hodgkin lymphoma.
 57. The method according to claim 53 wherein said myeloma is multiple myeloma.
 58. The method according to claim 53 wherein said antibody is an antibody having cytotoxicity.
 59. The method according to claim 58 wherein said cytotoxicity is ADCC activity.
 60. The method according to anyone of claims 53 to 59 wherein said antibody is a monoclonal antibody.
 61. The method according to claim 60 wherein said antibody is a chimeric antibody, a humanized antibody, or a human antibody.
 62. The method according to claim 60 wherein said antibody is an anti-HM1.24 antibody produced by a hybridoma having the Deposit No. FERM BP-5233.
 63. The method according to claim 61 wherein said chimeric antibody or humanized antibody is a chimeric antibody or humanized antibody produced by a hybridoma having the Deposit No. FERM BP-5233.
 64. A method of treating hematopoietic tumors comprising administering the expression-enhancing agent according to claim 23 and an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO:
 2. 65. A method of treating a hematopoietic tumor comprising administering the expression-enhancing agent according to claim 26 and an antibody that specifically binds to a protein containing the amino acid sequence set forth in SEQ ID NO:
 2. 